MBIO 3410 Lecture Notes - Lecture 2: Alkaline Phosphatase, Radionuclide, Fluorescence
24 Dec 2015
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Lecture 2b (cid:1) (cid:1) (cid:1) (cid:1) (cid:1) (cid:1) (cid:1) Incorporate modified nucleotides or molecules that fluoresce, generate biochemoluminescence, or emit particles (i. e. radioactive isotopes) Polynucleotide kinase (pnk) requires gamma atp32 and 5" ends that have been dephosphorylated via alkaline phosphatase. Damage dna randomly and repair with dna polymerase i. Dnase i nicks dna and dna polymerase repairs the damage. Add short oligonucleotides, dntps, modified nucleotide, buffer, and. Attach a fluorescent compound to dna that emits light of a visible wavelength upon being exposed to uv light. Attach a compound to dna for which an antibody exists that has an enzyme attached that catalyzes a reaction that releases light. Isotope generates decay emission (beta particles and/or gamma rays) These emissions will expose x-ray film and generate an image. Cross-link dna to membrane (via uv light or heat) Need sufficient rna to get a signal (not very sensitive) More sensitive methods include rt-pcr, rnaseq, and microarrays.
